Diabetes - especially in its type 2 form (T2D) - is one of the most potent risk factors for coronary artery disease (CAD). To decrease the cardiovascular burden of diabetes, a better understanding of the mechanisms linking the diabetic milieu to atherosclerosis is needed, so that effective interventions specifically targeted to diabetic subjects can be developed. Our strategy has been to seek this knowledge through genetic studies. Using a genome-wide approach, we have identified a genetic locus in the region of the glutamate-ammonia ligase (GLUL) gene on chromosome 1q25 that is associated with CAD (p= 2 x10-8) exclusively among diabetic subjects. The risk allele is also associated with reduced expression of the GLUL genes in endothelial cells and a decreased plasma pyroglutamic to glutamic acid ratio, suggesting mechanistic links with the ?-glutamyl cycle. Prompted by these findings, we have successfully piloted the use of metabolomics as an additional tool to dissect the pathways involved in the development of CAD in diabetes. Our goals are now: 1. To systematically assess the contribution of genetic variability in the glutamic and ?-glutamyl pathways to CAD risk in T2D. We will look for CAD-associated variants at key loci regulating glutamic acid metabolism and the ?-glutamyl cycle, focusing on low-frequency variants since these were not captured by the previous GWAS. Taking advantage of next-gen sequencing technology, we will comprehensively re-sequence these genes and all ENCODE-defined regulatory elements in a 500 Kb radius in over 5,000 CAD-positive cases and CAD- negative controls, all with T2D. We will seek replication of significant findings in 9,000 T2D subjects from additional case-control sets. 2. To gain insights into the metabolic mechanisms linking GLUL and other genes in the glutamic and ?-glutamyl pathways to CAD risk in T2D. We will investigate the hypothesis that the genetic effect is due to increased susceptibility to oxidative stress resulting from an impairment of the ?- glutamyl cycle and glutathione production. To this end, we will conduct targeted metabolomic studies of glutathione and oxidative stress markers in RBC and plasma samples from T2D patients and will relate these traits to the GLUL risk variants. Triangulation techniques will be employed to establish the causal path between genetic variants and oxidative stress.3. To identify other metabolic pathways, in addition to those regulated by the GLUL locus, that may influence cardiovascular risk in T2D. We will perform global metabolomic profiling of baseline plasma samples from a nested case-control study from a Joslin cohort, comparing subjects who experienced cardiovascular events during follow-up to those who did not. We will investigate the generalizability of positive findings in external validation studies. Supported by robust preliminary data and combining biased and unbiased approaches, this research will generate new knowledge that will be instrumental for the development of new interventions specifically directed at tackling CAD among diabetic patients.